Abstract The objective of this study was to compare the potential of mono‐rhamnolipids (mono‐RML) and di‐rhamnolipids (di‐RML) against biofilm formation on carbon steel coupons submitted to oil produced water for 14 days. The antibiofilm effect of the RML on the coupons was analyzed by scanning electron microscopy (SEM) and by epifluorescence microscopy, and the contact angle was measured using a goniometer. SEM analysis results showed that all RML congeners had effective antibiofilm action, as well as preliminary anticorrosion evaluation confirmed that all RML congeners prevented the metal deterioration. In more detail, epifluorescence microscopy showed that mono‐RML were the most efficient congeners in preventing microorganism's adherence on the carbon steel metal. Image analyses indicate the presence of 15.9%, 3.4%, and <0.1% of viable particles in di‐RML, mono/di‐RML and mono‐RML pretreatments, respectively, in comparison to control samples. Contact angle results showed that the crude carbon steel coupon presented hydrophobic character favoring hydrophobic molecules adhesion. We calculated the theoretical polarity of the RML congeners and verified that mono‐RML (log P = 3.63) presented the most hydrophobic character. This had perfect correspondence to contact angle results, since mono‐RML conditioning (58.2°) more significantly changed the contact angle compared with the conditioning with one of the most common surfactants used on oil industry (29.4°). Based on the results, it was concluded that rhamnolipids are efficient molecules to be used to avoid biofilm on carbon steel metal when submitted to oil produced water and that a higher proportion of mono‐rhamnolipids is more indicated for this application.