Abstract BACKGROUND There is a public interest in developing bio‐surfactants due to their low toxicity and high biodegradation potential. However, their biological, surface, and behavior to use with agrochemicals has not been investigated. RESULTS Critical micelle concentrations (CMCs) for the synthetic surfactant dioctyl sodium sulfosuccinate (DOSS), the bio‐surfactant rhamnolipid (RL), and the bio‐surfactant surfactin (SF) were 1200, 50, and 50 mg L −1 , respectively. Based on the ability of the surfactants to reduce the surface tension of trifloxysulfuron‐sodium spray solution at 0.25 to 1× CMC, they are ranked SF > RL > DOSS, while at 1.5 to 4× CMC, they are ranked SF = RL > DOSS. Without surfactant, trifloxysulfuron‐sodium at 10.04 g ha −1 reduced johnsongrass growth up to 50% (ED 50 ). At best, SF at 1 to 4× CMC halved ED 50 . Unlike DOSS, which increased ED 50 (12.89 g ha −1 ) due to a phytotoxic effect, SF and RL at 4× CMC decreased ED 50 (5.19 and 6.50 g ha −1 , respectively) without a phytotoxic effect. A 5‐μl droplet containing SF dried faster due to greater spread on the leaf surface than other surfactants. Although the wetted area of the leaf with the droplet containing RL was wider than that of DOSS, it took longer to dry. This observation contradicts the previous theory. CONCLUSION In terms of dosage, safety, and efficacy, the RL and SF were comparable to DOSS in other tank‐mix with trifloxysulfuron‐sodium. It seems that RL also works as a humectant, while SF likely works as a wax solubilisant. © 2023 Society of Chemical Industry.