A new strain, namely Lysinibacillus sp. BV152.1 was isolated from the rhizosphere of ground ivy ( Glechoma hederacea L.) producing metabolites with potent ability to inhibit biofilm formation of an important human pathogens Pseudomonas aeruginosa PAO1, Staphylococcus aureus, and Serratia marcescens. Structural characterization revealed di-rhamnolipids mixture containing rhamnose (Rha)-Rha-C10-C10, Rha-Rha-C8-C10, and Rha-Rha-C10-C12 in the ratio 7:2:1 as the active principle. Purified di-rhamnolipids, as well as commercially available di-rhamnolipids (Rha-Rha-C10-C10, 93%) were used as the substrate for the chemical derivatization for the first time, yielding three semi-synthetic amide derivatives, benzyl-, piperidine-, and morpholine. A comparative study of the anti-biofilm, antibacterial and cytotoxic properties revealed that di-Rha from Lysinibacillus sp. BV152.1 were more potent in biofilm inhibition, both cell adhesion and biofilm maturation, than commercial di-rhamnolipids inhibiting 50% of P. aeruginosa PAO1 biofilm formation at 50 μg mL -1 and 75 μg mL -1, respectively. None of the di-rhamnolipids exhibited antimicrobial properties at concentrations of up to 500 μg mL -1. Amide derivatization improved inhibition of biofilm formation and dispersion activities of di-rhamnolipids from both sources, with morpholine derivative being the most active causing more than 80% biofilm inhibition at concentrations 100 μg mL -1. Semi-synthetic amide derivatives showed increased antibacterial activity against S. aureus, and also showed higher cytotoxicity. Therefore, described di-rhamnolipids are potent anti-biofilm agents and the described approach can be seen as viable approach in reaching new rhamnolipid based derivatives with tailored biological properties.