The escalating prevalence of antibiotic resistance poses a substantial global health threat. While many antibiotics exhibit potent antimicrobial activity, their efficacy is often compromised by limitations in targeted delivery and biofilm penetration, potentially resulting in collateral tissue damage. Nano-particle-based antibiotic delivery systems offer a promising solution, leveraging their unique properties to facilitate precise targeting of infected sites, minimize systemic exposure, and enhance antibiotic efficacy against recalcitrant biofilms. In this study, we investigate the potential of incorporating small molecules with bacterial surface affinity to improve the targeting capabilities of nanoscale delivery systems. We synthesized various polymers using RAFT polymerization, incorporating different small molecules at varying ratios. These polymers self-assembled into nanoparticles and encapsulated a hydrophobic antibiotic, Rifampicin, which resulted in three Rifampicin-encapsulated nanoparticles: Rif@NP1, Rif@NP2, and Rif@NP3. The sizes of three nanoparticles ranged from 124.6 ± 31.1 to 239.7 ± 17.8 nm, and their ζ potential is from −45.4 ± 2.5 nm to −22.3 ± 1.8 nm. The release of Rifampicin from nanoparticles was monitored in PBS buffer at pH 6.0 and pH 7.4. While Rif@NP1 released less than 20% of Rifampicin in both pH, Rif@NP2 and Rif@NP3 were able to release up to 40% after 72 h. The antimicrobial activity of three nanoparticles was tested on Escherichia coli and Staphylococcus aureus, and Rif@NP3 showed a better effect on both bacteria than the other nanoparticles. In conclusion, three polymer-based nanoparticles encapsulating Rifampicin, although three nanoparticles possess different properties, were able to release Rifampicin and show antimicrobial effects on E. coli and S. aureus, which indicated the potential to be employed in further studies such as biofilm-forming prevention.