New anti-HIV-1 drugs that target different viral proteins or genes at various steps in the viral life cycle are highly expected. HIV-1 assembly and disassembly (uncoating) processes are critical for the HIV-1 replication. HIV-1 capsid (CA) and human cyclophilin A (CypA) play essential roles in these processes. Using an in vitro screening system, we evaluated 52 thiourea derivatives for their potential CA and CypA-inhibiting activities. The antiviral activity of these compounds is correlated with their CA assembly inhibitory ability and with their anti-PPIase activity, suggesting that these compounds could block HIV-1 replication by disrupting CA assembly and inhibiting the PPIase activity of CypA to interfere with capsid disassembly. Among them, three compounds D4, D5, and D6 displayed the most promising potency with CA-assembly rate 15.78, 18.42, and 7.97(×10⁻⁵) OD/s, and their IC₅₀ for inhibition of PPIase activity 0.45, 0.65, and 0.33 μM, respectively. The potent protein inhibitory activity resulted in their very low EC₅₀ values (≤1.00 μM). They can be used for rational design of novel anti-HIV-1 drugs.