Viral infections have been appointed as the main component of environmental susceptibility to multiple sclerosis (MS). Interferon beta is an immunomudulatory treatment that is able to modify the natural course of the disease; nonetheless, its mechanism of action in not well established yet. The objectives of the present study were (1) to evaluate the bioavailability of interferon beta through the measurement of the expression of myxovirus resistance protein (MxA), metalloproteinase 9 (MMP-9), and its inhibitor (TIMP-1); (2) to analyze its antiviral efficiency through the measurement of human herpesvirus-6 (HHV-6) prevalence; and (3) to correlate both parameters (bioavailability and antiviral efficiency) with the relapse rate in multiple sclerosis (MS) patients treated with interferon beta. Pairs of blood and serum samples were collected from 54 MS patients during five visits in 1 year: one before the start of the treatment and four during interferon beta treatment. Expression of MxA, MMP-9, and TIMP-1 was analyzed by quatitative real-time polymerase chain reaction (qRT-PCR) and HHV-6 genomes were detected by qPCR. The results showed a correlation between MxA and relapse rate (P = .014). MMP-9/TIMP-1 ratio was increased among the patients with relapses, and decreased among the relapse-free patients, although differences were not statistically significant. Furthermore, our results suggest a possible role for HHV-6 in MS, because 42.8% of patients with viral reactivations experienced at least one relapse versus 22.5% of patients without viral reactivations. Lastly, regarding the antiviral effectiveness of the interferon beta, the HHV-6 prevalence decreased from 58% to 36% in PBMCs and from 18.5% to 12.2% in sera; furthermore, a good correlation with the bioavailability of interferon beta was found, because patients with a decrease in HHV-6 prevalence had higher levels of MxA (P = .046, at the third month).