Chemotherapy for cancer could have negative effects on HIV-1 dynamic in addition to the effects on immunological status. At the moment few data are available about the effects of chemotherapy on systemic HIV-1 replication, but the effects on the central nervous system, considered an independent compartment for viral replication, has never been investigated. We studied 19 HIV-1-infected patients with non-Hodgkin lymphoma (NHL) treated concomitantly with chemotherapy and highly active antiretroviral therapy (HAART) to evaluate HIV-1 replication and assess virological response to HAART in cerebrospinal fluid during chemotherapy. No patients were diagnosed with lymphoma involvement of the central nervous system. In 18 of 19 patients an HIV-1 load below 200 copies/ml was obtained in cerebrospinal fluid (CSF) during treatment. A correlation between plasma and CSF HIV-1 RNA levels was present at baseline, and was confirmed at the fourth cycle of chemotherapy. A significant decline in the mean difference between plasma and CSF HIV-1 RNA load was observed when comparing the value at the first cycle of chemotherapy with subsequent cycles. HAART even during chemotherapy allows us to obtain an effective control of HIV-1 infection in CSF in patients affected by NHL. In HIV-infected patients with NHL, the contemporaneous administration of HAART and chemotherapy is advisable to obtain a suppression of HIV-1 replication in CNS compartment during the potentially immunosuppressing effect of cancer treatment.