Abstract Rhamnolipids (RMLs) have more effectiveness for specific uses according to their homologue proportions. Thus, the novelty of this work was to compare mono‐RMLs and di‐RMLs physicochemical properties on microbial enhanced oil recovery (MEOR) applications. For this, RML produced by three strains of Pseudomonas aeruginosa containing different homologues proportion were used: a mainly mono‐RMLs producer (mono‐RMLs); a mainly di‐RMLs producer (di‐RMLs), and the other one that produces relatively balanced amounts of mono‐RML and di‐RML homologues (mono/di‐RML). For mono‐RML, the most abundant molecules were Rha‐C 10 C 10 (m/z 503.3), for di‐RML were RhaRha‐C 10 C 10 (m/z 649.4) and for Mono/di‐RML were Rha‐C 10 C 10 (m/z 503.3) and RhaRha‐C 10 C 10 (m/z 649.4). All RMLs types presented robustness under high temperature and variation of salinity and pH, and high ability for oil displacement, foam stability, wettability reversal and were classified as safe for environment according to the European Union Directive No. 67/548/EEC. For all these properties, it was observed a highlight for mono‐RML. Mono‐RML presented the lowest surface tension (26.40 mN/m), interfacial tension (1.14 mN/m), and critical micellar concentration (CMC 27.04 mg/L), the highest emulsification index (EI 24 100%) and the best wettability reversal (100% with 25 ppm). In addition, mono‐RML showed the best acute toxicity value (454 mg/L), making its application potential even more attractive. Based on the results, it was concluded that all RMLs homologues studied have potential for MEOR applications. However, results showed that mono‐RML stood out and have the best mechanism of oil incorporation in micelles due their most effective surface‐active physicochemical features.