Tomato brown rugose fruit virus (ToBRFV) is considered an emerging disease and a viral pandemic for tomato consumers. The objectives of this research were to analyze the biological and physicochemical characteristics of ToBRFV in tomato and tobacco plants, as well as to evaluate its natural host range. Inoculant seeds were recovered from ToBRFV-infected tomato samples in Coahuila, Mexico, and confirmed by RT-PCR. In the first greenhouse experiment, tomato plants of the F1 hybrid variety 172–300 (Yüksel), infected with ToBRFV, were used to evaluate viral inclusions (VI), dilution endpoint (DEP), the incubation period (IP), and latency period (LP). In a greenhouse experiment, Nicotiana longiflora plants were inoculated with ToBRFV to determine the in vitro longevity (IVL) and thermal inactivation (TI) of the virus in sap. Finally, the inoculation of tomato plants grown in open fields was carried out to evaluate transmission to natural hosts. The plants tested for possible ToBRFV reservoirs near the inoculum source were inspected and confirmed by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The results indicate that the VIs on tomato leaves manifested as X-bodies and rounded, stacked plaques within epidermal cells. The DEP required to induce the infection in plants was from a ToBRFV concentration of 1 × 10−5.5, the IP of ToBRFV occurred 9 to 12 days post-inoculation, and LP could be detected one day after inoculation. The TI of ToBRFV in N. longiflora plants occurred at 85 °C for 30 min. Ipomoea purpurea, Mirabilis jalapa, Clematis drummondii, and Solanum tuberosum were newly identified hosts of ToBRFV. The results found contribute to a better understanding of the impact of ToBRFV, managing and preventing the spread of ToBRFV in diverse environments.