In the past, the response of microbial populations to anthropogenic disturbances was studied using conventional methods based on cultivation of microorganisms and on measurement of their metabolic activities (Fantroussi et al., 1999). However, these culturing methods often account for a small proportion of the total microbial community (Ibekwe and Kennedy, 1998; Hill et al., 2000). To overcome this, molecular techniques were developed and these allowed for the analyses of microorganisms in their natural habitats. Analysis of the 16S rRNA molecule and its corresponding gene (16S rDNA) has been the most widely used approach in the last decade (Amman et al., 1995). Although molecular techniques based on PCR have been used to eliminate the bias of culturing methods, they also have their drawbacks (Wintzingerode et al., 1997; Kirk et al., 2004). As another alternative, Garland and Mills (1991) developed a rapid community-level physiological approach to study microbial communities. The use of the community-level approach to microorganisms provided an accurate and meaningful measure of the heterotrophic microbial community by measuring the community’s metabolic abilities (Garland and Mills, 1991). Zak et al. (1994) used the method to study the functional diversity of microbial communities. The approach has been used to study the soil functional diversities in polluted or disturbed environments. Over the years, the application of gypsum in agriculture has received much attention. The gypsum has been used to ameliorate both acidic and alkali soils with elevated amounts of salinity (Suhayda et al., 1997; Sun et al., 2000). In these studies, the application of gypsum lead to changes in the soil chemical properties by causing a drastic increase in the amount of exchangeable calcium and sulphate and reduced the levels of exchangeable aluminium. It has been noted that high levels of aluminium and/or reduced amounts of calcium restrict root elongation and thus hindered the plants ability to access adequate water (Sun et al., 2000). Also, the replacement of sodium ions with calcium ions resulted in the flocculation of soil particles and improved the porous structure and water permeability of the soil (Suhayda et al., 1997). This study revealed that the application of the gypsiferous mine water did not have any negative impact on the bacterial communities. In fact, on average, the bacterial diversities were found to be higher in the gypsum-irrigated soils. This was most evident in pivot Major and Tweefontein, where the gypsum-irrigated soils were more diverse than the control soils. DGGE results from pivot Major and Tweefontein revealed a high level of bacterial diversity in gypsum-irrigated soils, as estimated by the number of dominant bands. Also, the number of heterotrophic bacteria in the gypsum-irrigated soils was one to two orders of magnitude higher than in the control soils. Principal component analysis performed on BIOLOG data showed that in both pivot Major and Tweefontein, the gypsum-irrigated soils were able to utilise a wider range of carbon sources as compared to their control counterparts. The bacterial communities in pivot Four appeared to be steady in both the gypsum-irrigated soils and the control soils. The number of visible DGGE bands was consistent between the gypsum-irrigated and the control soils. The heterotrophic bacterial counts in the gypsum-irrigated soils had an average of 273x106 cfu g-1 soil and those present in the control soils were slightly higher at 380x106 cfu g-1 soil. Principal component analysis revealed no differences in terms of substrate utilisation capabilities among the gypsum-irrigated soils and the control soils. All three techniques revealed no significant difference in community structures between soil profiles at 0-10 cm and 40-60 cm. The lack of difference could be attributed to the crops planted in all three pivots during sampling. The root system of Zea Maysplants enhanced microbial growth by exuding nutrients such as amino acids and sugars. In conclusion, the application of polyphasic approach proved successful in studying the response of soil bacterial communities to gypsiferous mine water. The use of both culture-dependent and culture-independent methods is recommended as the methods compensate each other’s limitations and therefore provide a more detailed description of the community. In this study, the application of gypsiferous mine water did not have an adverse effect on the soil bacterial communities. In fact, the addition of gypsiferous mine water seemed to ameliorate the soil bacterial communities. However, further comprehensive study is needed to determine the response of bacterial communities to gypsiferous mine water over longer periods of time. 16S rDNA sequencing and analysis of DGGE bands should also be done to identify the bacterial species present in the gypsum-irrigated samples.