Biosurfactants play a very important role in the fresh produce industry, especially as anti-microbial agents. This study aimed to investigate the anti-oxidant and anti-bacterial activities of a glycolipoprotein biosurfactant (BSF) produced by Lactobacillus plantarum MGL-8, as well as its anti-bacterial mechanism of action against Listeria monocytogenes DMST17303. Its application as a sanitizer for fresh Chinese kale was investigated at different storage temperatures and exposure times. Its safety was monitored by cytotoxic effect to human skin HaCaT keratinocyte cells. The BSF exhibited high anti-oxidant capacities, with ferric-reducing from the FRAP assay of 15.96 μgVCEA/mg and ABTS scavenging activities with an IC50 of 877.00 μg/mL. BSF demonstrated anti-bacterial activity against L. monocytogenes through decreasing the fluidity and increasing permeability of the cell membrane, resulting in reducing membrane function and leakage of intracellular components. The study revealed that the appropriate washing procedures of W2 (BSF 400 μg/mL for 20 min) and W3 (BSF 450 μg/mL for 10 min) efficiently inhibited the growth of L. monocytogenes in fresh Chinese kale stored at 5 °C for 7 and 14 days. BSF did not exhibit any cytotoxic effects on HaCaT cells at concentrations of up to 1,000 μg/mL, suggesting using BSF for disinfect L. monocytogenes in fresh Chinese kale at 450 μg/mL is safe for human. Therefore, it is recommended to wash fresh Chinese kale with BSF at 400 or 450 μg/mL for 20 or 10 min, respectively, to reduce food safety risks such as L. monocytogenes before storing at 5 °C for 14 days.