Background Bovine herpesvirus type 5 is an important agent of meningoencephalitis in cattle and has been identified in outbreaks of bovine neurological disease in several Brazilian states. In recent years, oxoquinoline derivatives have become an important focus in antiviral drug research. Methods The cytotoxicity and anti BoHV-5RJ42/01 activity of a set of synthetic 4-oxoquinoline derivatives 4a-k were assayed on Madin–Darby Bovine Kidney cell and antiviral activity by plaque reduction assay. Results: The most promising substance (4h) exhibited CC₅₀ and EC₅₀ values of 1,239 μM ±5.5 and 6.0 μM ±1.5, respectively, with an SI =206. Two other compounds 4j (CC₅₀ = 35 μM ±2 and EC₅₀ = 24 μM ±7.0) and 4k (CC₅₀= 55 μM ±2 and EC₅₀ = 24 μM ±5.1) presented similar inhibitory profile and selectivity indexes of 1.4 and 2.9, respectively. The results of the time-of-addition studies revealed expressive reduction of virus production (≥80%) in different stages of virus replication cycle except for compound 4h that slightly inhibited virus yield in the first 2 h post infection, but it showed expressive virus inhibition after this time. Conclusions All three compounds slightly interact with the virus on the virucidal assay and they are not able to block virus attachment and penetration. Antiviral effect of oxoquinoline 4h was more prominent than acyclovir which leads us to suggest compound 4h as a promising molecule for further anti-BoHV-5 drug design.