Secondary soil salinilization is a big problem in irrigated agriculture. We have studied the effects of irrigation-induced salinity on microbial biomass of soil under traditional cotton (Gossypium hirsutum L.) monoculture in Sayhunobod district of the Syr-Darya province of northwest Uzbekistan. Composite samples were randomly collected at 0–30 cm depth from weakly saline (2.3 ± 0.3 dS m−1), moderately saline (5.6 ± 0.6 dS m−1), and strongly saline (7.1 ± 0.6 dS m−1) replicated fields, 2-mm sieved, and analyzed for pH, electrical conductivity, total C, organic C (COrg), and extractable C, total N and P, and exchangeable ions (Ca2+, Mg2+, K+, Na+, Cl−, and CO32−), microbial biomass (Cmic). The Na+ and Cl− concentrations were 36-80% higher in strongly saline compared to weakly saline soil. The COrg concentration was decreased by 10% and CExt by 40% by increasing soil salinity, whereas decrease in Cmic ranged from 18-42% and the percentage of COrg present as Cmic from 8% to 26%. We conclude that irrigation-induced secondary salinity significantly affects soil chemical properties and the size of soil microflora.