Multidrug membrane transporters exist in both prokaryotic and eukaryotic cells and cause multidrug resistance (MDR), which results in an urgent need for new and more effective therapeutic agents. In this study, we used three different sized antibiotic nanocarriers to study their mode of action and their size-dependent inhibitory effects against Escherichia coli (E. coli). Antibiotic nanocarriers (AgMUNH–Oflx NPs) with 8.6 × 10², 9.4 × 10³ and 6.5 × 10⁵ Oflx molecules per nanoparticle (NP) were prepared by functionalizing Ag NPs (2.4 ± 0.7, 13.0 ± 3.1 and 92.6 ± 4.4 nm) with a monolayer of 11-amino-1-undecanethiol (MUNH₂) and covalently linking ofloxacin (Oflx) with the amine group of AgMUNH₂ NPs, respectively. We designed a modified cell culture medium for nanocarriers to be stable (non-aggregated) over 18 h of cell culture, which enabled us to quantitatively study their size and dose dependent inhibitory effects against E. coli. We found that the inhibitory effects of Oflx against E. coli highly depended upon the dose of Oflx and the size of the nanocarriers, showing that an equal amount of Oflx that was delivered by the largest nanocarriers (92.6 ± 4.4 nm) were most potent with the lowest minimum inhibitory concentration (MIC₅₀) and created the longest and highest percentage of filamentous cells, while the smallest nanocarriers (2.4 ± 0.7) were least potent with the highest MIC₅₀ and produced the shortest and lowest percentage of filamentous cells. Interestingly, the same amount of Oflx on 2.4 ± 0.7 nm nanocarriers showed a 2× higher MIC and created 2× shorter filamentous cells than free Oflx, while the Oflx on 13.0 ± 3.1 and 92.6 ± 4.4 nm nanocarriers exhibited 2× and 6× lower MICs, and produced 2× and 3× longer filamentous cells than free Oflx, respectively. Notably, the three different sized AgMUNH₂ NPs (absence of Oflx) showed negligible inhibitory effects and did not create filamentous cells. The results show that the filamentation of E. coli highly depends upon the sizes of nanocarriers, which leads to the size-dependent inhibitory effects of nanocarriers against E. coli.