The increased use of plastic films and pesticides on agricultural soil leads to the accumulation of plastic debris and pesticide residues in soil. This accumulation has become a serious environmental issue, as it threatens life of earthworms, inhibits the enzyme activities and microbial diversity, and contributes to the loss of soil microbial carbon and nitrogen. However, little information is available regarding the effects of pesticides on soil dissolved organic matter (DOM). It is also unknown how plastic debris, especially small-sized particles called microplastics, influences the effects of pesticides on soil DOM. In this study, we performed a 30-day soil incubation experiment. Three levels of the common herbicide glyphosate were applied to soil: 0 (control, CK), 3.6 kg ha− 1 (G1) and 7.2 kg ha− 1 (G2). We also tested four levels of glyphosate and microplastics (homopolymer polypropylene powder) co-addition: 3.6 kg ha− 1 + 7% (w/w) (M1G1), 3.6 kg ha− 1 + 28% (w/w) (M2G1), 7.2 kg ha− 1 + 7% (w/w) (M1G2), and 7.2 kg ha− 1 + 28% (w/w) (M2G2). Glyphosate addition slightly increased soil fluorescein diacetate hydrolase (FDAse) and phenol oxidase (PO) activities. Although the glyphosate addition significantly promoted the accumulation of dissolved organic phosphorus (DOP) within the first 14 days, the M2 treatment decreased DOP at day 30. M2G1 and M2G2 increased soil FDAse activity and promoted the accumulation of DOC and DOP relative to G1 and G2 respectively while M1G1 and M1G2 benefited DON accumulation. Our results highlighted that the interaction between glyphosate and low microplastics content negatively affected DOC and DOP dynamics, leading to the loss of bioavailable C and P loss. The interaction between glyphosate and high content microplastics negatively affected DON compared with glyphosate addition, possibly decreasing DON.