PurposeAntibiotic resistance is a huge problem that stays to challenge the healthcare sector in a large part of the world in both developing and developed countries. The spread of multi drug resistant (MDR) bacteria in hospital and community settings remains a widely uncertain problem and a heavy burden to health services. MethodsThis study unveils the in vitro and in vivo anti-ESBL potential of Methyl oleate (MO) and Palmitic acid (PA) against ESBL producing MDR bacterial pathogens such as Escherichia coli and Klebsiella pneumoniae. Microscopic observations unveiled the anti-ESBL efficacy of test compounds. MTT assay, in vivo anti-infective efficiency of MO and PA was tested with different concentrations. ResultsThe pure compounds of MO and PA from Oxynema thaianum demonstrated high inhibitory activity in MIC and MBC assays against MDR E. coli and K. pneumoniae. Moreover, the anti-ESBL potential of MO and PA was validated through light, confocal laser scanning and scanning electron microscopic analyses. The IC50 values of MO and PA against A549 cells was recorded as 625 μg mL−1 and 514 μg mL−1, respectively. In Artemia nauplii cytotoxicity assay, the LC50 value of MO and PA were recorded as 53.33 μg mL−1 and 50 μg mL−1 respectively. The 96 h lethal concentrations obtained for Lobeo rohita treated with different concentrations of Methyl oleate and Palmitic acid. The LC50 for MO and PA was 50 mg L−1 and 100 mg L−1, respectively. ConclusionTherefore the study concluded that the promising effects of MO and PA can be used as an alternative biological agent which could be positively explored to treat ESBL producing MDR pathogens.