Intercropping mulberry and alfalfa in the field experiment increased enzyme activities in the soil compared to monoculture. Invertase and peroxidase activities were higher in intercropped alfalfa, while acid phosphatase activities were higher in intercropped mulberry and alfalfa. The diversity of soil microbial communities was also affected, with higher diversity in intercropped mulberry and lower diversity in intercropped alfalfa. The main carbon sources for soil microbes differed between intercropped and monoculture systems, with intercropped alfalfa showing a shift towards amino acids and polymers. Intercropping patterns had varying effects on soil enzyme activities.