Mycoplasma (M.) hyopneumoniae, the etiological agent of swine enzootic pneumonia, has been reported to increase the susceptibility to secondary infections and modulate the respiratory microbiota in infected pigs. However, no studies have assessed the influence of M. hyopneumoniae on the respiratory microbiota diversity under experimental conditions. Therefore, this study evaluated the impact of M. hyopneumoniae infection on the respiratory microbiota of experimentally infected swine over time. To accomplish this, 12 weaned pigs from a M. hyopneumoniae-free farm were divided into two groups: M. hyopneumoniae strain 232 infected (n = 8) and non-infected (n = 4). The first group received 10 mL of Friis medium containing 107 CCU/mL of M. hyopneumoniae while the control group received 10 mL of sterile Friis medium. Inoculation of both groups was performed intratracheally when the animals were 35 days old (d0). At 28 days post-inoculation (dpi) and 56 dpi, 4 infected animals plus 2 controls were humanely euthanized, and biopsy samples of nasal turbinates (NT) and bronchus-alveolar lavage fluid (BALF) samples were collected. The DNA was extracted from the individual samples, and each group had the samples pooled and submitted to next-generation sequencing. Taxonomic analysis, alpha and beta diversity indexes, weighted unifrac, and unweighted unifrac distances were calculated. A high relative frequency (99%) of M. hyopneumoniae in BALF samples from infected animals was observed with no significant variation between time points. The infection did not seem to alter the diversity and evenness of bacterial communities in NT, thus, M. hyopneumoniae relative frequency was low in NT pools from infected animals (28 dpi-0.83%; 56 dpi-0.89%). PCoA diagrams showed that BALF samples from infected pigs were grouped and far from the control samples, whereas NT from infected animals were not separated from the control. Under the present coditions, M. hyopneumoniae infection influenced the lower respiratory microbiota, which could contribute to the increased susceptibility of infected animals to respiratory infections.