Irrigation with saline water is a possible solution to alleviate freshwater shortages. The long-term use of saline water for irrigation requires consideration of the influence of salt on the environmental conditions of the soil. The objective of this field study was to determine the effects of three continuous years of saline water irrigation on physiochemical properties and microbial communities in drip-irrigated cotton fields. The three total dissolved solid (TDS) levels of irrigation water treatments were (i) 1 g L−1 (fresh water, FWI), (ii) 3 g L−1 (brackish water, BWI), and (iii) 7 g L−1 (salt water, SWI). After three years, the electrical conductivity (EC), sodium adsorption ratio (SAR), and contents of K+, Na+, Mg2+, Cl−, and SO42− in the SWI treatment were significantly higher than those in the FWI and BWI treatments, but there were no significant differences in EC and K+ between the FWI and BWI treatments. BWI treatment significantly increased microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), urease, and sucrase contents. The diversity and abundance of bacteria and fungi were not affected by saline water irrigation, but the microbial community structure was altered. Saline water irrigation resulted in an elevation in the bacterial abundance of the phylum Chloroflexi and a decline in Proteobacteria and Actinobacteria. For fungi, the abundance of the phylum Ascomycota in the BWI treatment was greater than that in the FWI and SWI treatments. Linear discriminant analysis effect size (NMDS) results indicated clear variation in the microbiota profiles between the FWI, BWI, and SWI treatments for bacteria. Regarding the fungal microbiota profiles, the BWI and SWI treatments had similar microbiota profiles but were different from the FWI treatment. The number of bacterial biomarkers gradually increased with increasing total dissolved solids of irrigation water, while the number of fungal biomarkers gradually decreased. Additionally, cotton yield was significantly and positively correlated with the observed species of fungi, while it was significantly and negatively correlated with EC. Redundancy analysis (RDA) showed that bacterial community structure was regulated by SAR and fungal community structure was regulated by soil salinity and bulk density (BD). Future research will need to look into how the structure of the microbial community and the associated functional microorganisms are gradually changing with increased irrigation frequency under saline irrigation, as well as explore and screen for advantageous functional microorganisms.