The utilization of bioherbicides in place of chemical herbicides has been described as one of the sustainable eco-friendly technologies that can improve agricultural outputs. This work investigated the impact of different carbon and nitrogen sources on the bioherbicidal efficiency of Lasiodiplodia pseudotheobromae (C1136). The preliminary screening of various carbon and nitrogen sources was carried out, out of which sucrose and peptone were selected for further C:N ratio investigations using strain C1136. The fungus produces a different quantity of mycelium biomass and spores in varied ratios of C:N when compared to the basal medium. Also, the effect of C:N ratio was tested by evaluating for leaf necrosis assay, mortality rate and the dry-matter biomass from the aerial parts of Chromolaena odoratum and Echinochloa crus-galli under guided laboratory and screen house conditions. Both the 7.5:1 and 10:1 ratios produced optimal spores count of 6.1 × 107 and 5.9 × 107 CFU/mL, respectively (p = 0.05) when compared to the control containing potatoes dextrose broth without any carbon or nitrogen amendment produced a spores count of 1.1 × 107 CFU/mL. Also, the phytotoxic metabolite amended with C:N of 10:1 showed a strong herbicidal, high mortality and a significant decrease in dry weight on the seedling on the tested weeds. This demonstrated hypothetically the optimization influence of carbon\ (sucrose)–nitrogen (peptone) manipulations either in variable combination or as single nutrient based on the cultural development of strain C1136. Spores, mycelium biomass and biocontrol efficacy of the fungus all experienced accretion in sucrose and peptone nutrient environment and likewise support their use as an attractive alternative in the optimization of mycoherbicidal assays.