Current solutions for bacteria and viruses identification are based on time-consuming technics with complex preparation procedures. In the present work, we revealed label-free the presence of free viral particles and bacteria with a computational two-photon fluorescence (C-TPF) strategy. Six bacteria were tested: Escherichia coli, Staphylococcus epidermidis, Proteus vulgaris, Pseudomonas fluorescens, Bacillus subtilis and Clostridium perfringens. The two families of viral particles were the herpes virus with the cytomegalovirus (CMV, 300 nm of diameter) and the coronavirus with the SARS-CoV-2 (100 nm of diameter). The instrumental and computational pipeline FAMOUS optimized the produced 3D images. The origin of the fluorescence emission was discussed for bacteria regarding to their two-photon excitation spectra and attributed to the metabolic indicators (FAD and NADH). The optical and computational strategy constitute a new approach for imaging label-free viral particles and bacteria and paves the way to a new understanding of viral or bacterial ways of infection.