In the field of nanomedicine, nanoparticles are developed to target antibiotics to sites of bacterial infection thus enabling adequate drug exposure and decrease development of resistant bacteria. In the present study, we investigated the encapsulation of two antibiotics with different polarity into different PEGylated polymeric nanoparticles based on aliphatic polyesters, to obtain a better understanding of critical factors determining encapsulation and release. The nanoparticles were prepared from diblock copolymers comprising of a poly(ethylene glycol) block attached to an aliphatic polyester block of varying polarity: poly(lactic-co-glycolic acid) (mPEG-PLGA), poly(lactic-co-hydroxymethyl glycolic acid) (mPEG-PLHMGA) and poly(lactic-co-benzyloxymethyl glycolic acid) (mPEG-PLBMGA). Hydrophobic bedaquiline and hydrophilic vancomycin were encapsulated via single and double-emulsion solvent evaporation techniques, respectively. Encapsulation, degradation and release studies at physiological simulating conditions were performed. Drug polarity and preparation techniques influenced encapsulation efficiency into polymer nanoparticles, giving almost complete encapsulation of bedaquiline and approx. 30% for vancomycin independent of the polymer type. The nonpolar bedaquiline showed a predominantly diffusion-controlled release independent of polymer composition. However, polar vancomycin was released by a combination of diffusion and polymer degradation, which was significantly affected by polymer composition, the most hydrophilic polymer displaying the fastest release.