A series of novel 6-chloro-3-methyl-3,4-dihydroquinoxalin-2(1H)-ones bearing a 2,5-disubstituted thiophen-3-ylsulfonyl moiety were designed and synthesized aiming to improve the anti-HIV-1 activity of quinoxalinone derivatives. The target compounds were synthesized via microwave irradiation, Suzuki coupling and other reactions by modifying the 2- and 5-positions of the thiophene ring. Their anti-HIV-1 replication activities were evaluated using a cell-based model, and molecular docking with CDOCKER was performed to analyze the structure-activity relationships (SAR). Compounds 1b (with 5-chloro-2-methoxycarbonyl thiophene fragment) and 1d (with 5-iodo-2-methoxycarbonyl thiophene fragment) were identified as the most potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) with IC50 values at the 10⁻⁸ mol/L level. SAR analysis showed that halogen substituents (chloro, iodo) at the 5-position of the thiophene ring were favorable for activity, while bulky aromatic groups or hydrophilic substituents (e.g., carboxylic, hydroxymethyl, aldehyde) reduced activity. However, none of the tested compounds effectively inhibited the HIVRT-K103N and HIVRT-Y181C resistant strains. In summary, these quinoxalinone derivatives are potential NNRTIs, and the SAR results will facilitate the further development of novel potent quinoxaline-based NNRTIs.