Hudaira Drain of Pakistan is one of the largest storm water drains which is a potential threat to the environment and biodiversity. It is considered as the second largest water polluting body in Pakistan. The major polluting contributors are toxic industrial wastes, chemical fertilizers and poultry or animal waste. The objective of this study was to analyze the microbial diversity of wastewater from Hudiara Drain using culture-independent and culture-dependent techniques. For culture-independent analysis, the 16S rRNA gene-based Illumina sequencing technique was used. Metagenomic sequence analysis revealed a total of 20 different phyla in the source drain water samples. Bacterial phyla Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes, Acidobacteria, Chloroflexi, Planctomycetes, and Fusobacteria and archaeal phylum Thaumarchaeota were commonly identified from all the wastewater samples. A total of 282 genera of bacteria and archaea were identified in this study, with Bacillus, Nitrospira, Nitrososphaera, Gemmatimonas, Pseudomonas, Stenotrophomonas, Myxococcus, Azospirilum, Skermanella, Nitrososphaera, Pontibacter and Aquicella being the dominant genera. Using culture-dependent techniques, 32 bacterial strains were identified and characterized for heavy metal and antibiotic resistance. The common genera were Bacillus, Aeromonas and Enterobacter. These results suggest that the microbial diversity from Hudiara Drain wastewater samples is a potential source of clinically and industrially important bacterial and archaeal strains.