A field trial was conducted to investigate the ‘Phytotoxicity of pendimethalin on the emergence of seedlings in direct seeded rice (DSR) across different concentrations’ during the Kharif season of 2022 at the experimental farm of the School of Agriculture, Lovely Professional University, Phagwara, Punjab. The experiment was conducted using a randomized block design with five treatments using different doses of pendimethalin (1600, 1800, 2000, and 2200 gram active ingredient per hectare (g ai per ha), and control (untreated)). Application of pendimethalin at 1600 g ai per ha resulted in a significantly higher number of seedlings than other treatment groups. The lowest number of seedlings was observed in the 2200 g ai per ha treatment group. The number of microbes present was optimal during the treatment with 1600 g ai per ha pendimethalin. With an increase in the dosage of pendimethalin, a decrease in the count of soil microbes was observed, with the lowest microbial count observed with the highest dose of pendimethalin, i.e., 2200 g ai per ha. No phytotoxic effect was observed in the DSR treated with pendimethalin at 1600 g ai per ha. Therefore the present study has to be obtain the appropriate use of dosage to inhibit the toxicity of herbicide as well as maintain the soil fertility and microbial growth.