We have collaborated with the group of Prof. Maciej Guzik at Polish Academy of Sciences in this work on methods of obtaining various fatty acid esters (SFAEs) using combined biological, chemical and enzymatic methods. It presents how modification of SFAE’s hydrophobic chains can influence their cytotoxicity against human skin melanoma and prostate cancer cell lines. The compound’s cytotoxicity was determined by an MTT assay, which followed an assessment of SFAEs’ potential metastatic properties in concentrations below IC50 values. Despite relatively high IC50 values (63.3–1737.6 μM) of the newly synthesized SFAE, they can compete with other sugar esters already described in the literature. The chosen bioactives caused low polymerization of microtubules and the depolymerization of actin filaments in nontoxic levels, which suggest an apoptotic rather than metastatic process. Altogether, cancer cells showed no propensity for metastasis after treating them with SFAE. They confirmed that lactose-based compounds seem the most promising surfactants among tested sugar esters. This manuscript creates a benchmark for creation of novel anticancer agents based on 3-hydroxylated fatty acids of bacterial origin.