A total of 1.3 million people died from tuberculosis (TB) in 2022 (including 167,000 individuals with HIV) with a large proportion of them from sub-Saharan Africa. Unfortunately, there is paucity of data on influenza virus in both tuberculosis and HIV patients in Nigeria. Hence this study was designed to investigate influenza virus in tuberculosis and HIV patients attending chest clinics in Lagos State. A total number of 400 samples were collected from four different health facilities including three general hospitals and one tertiary institution health center. The population consists of 32.8% (131/400) males and 67.2% (269/400) females of which 11% (30/269) were pregnant. Their age ranged from 7 to > 65 years. Nasopharyngeal samples were extracted for influenza RNA with QIAamp mini kit followed by RT-PCR amplification. TB and HIV tests were performed as part of clinical management. Of the total patients tested, 26.8% (90/336) were HIV positive, 7.3% (29/400) had malaria parasitaemia, 2.5% (10/400) had tuberculosis and 1.8% (7/400) was asthmatic. Furthermore, the result showed 2.3% (9/400) low molecular prevalence to influenza A virus out of which 66.7% (6/9) were females. Interestingly, out of the nine (9) influenza A virus positive patients, 66.7% (6/9) had HIV, 44.4% (4/9) had TB while 33.3% (3/9) were co-infected with TB and HIV. Ikorodu General Hospital accounted for 55.6% (5/9) molecular prevalence to influenza while 44.4% (4/9) was recorded in Badagry General Hospital. In conclusion, this study is the first report of molecular detection of influenza virus in TB and HIV patients in Nigeria. The co-infection of influenza virus in TB and HIV patients underscores the need for a national surveillance and provision of influenza vaccines and antiviral drugs for the affected individuals.