Early events occurring after inoculation of Pinus radiata seedlings with Macrophomina phaseolina, fungal pathogen causing charcoal root rot, are unknown. The objectives of this study were to determine the germination process of microsclerotia and penetration of hyphae of M. phaseolina into roots of P. radiata seedlings and to establish probably effects on photosynthetic efficiency during early hours of infection pathogen. For this, 25-day-old seedling roots were inoculated with M. phaseolina microesclerotia under in vitro conditions. First hours of disease cycle after root inoculation were studied by scanning electron microscopy and efficiency of photosystem II by measuring the fluorescence of chlorophyll a, after inoculating of P. radiata seedling roots with M. phaseolina microesclerotia. The penetration of the pathogen into the root occurred 48 h post inoculation (hpi), and surface colonization was evident by the presence of numerous hyphae after 72 hpi. The parameters of chlorophyll a fluorescence showed variations at once occurred colonization and penetration of seedling roots, with a decrease of Fv/Fm values from 120 hpi. Infection root of P. radiata with M. phaseolina microesclerotia generated a stress in the plant which resulted in variations in fluorescence parameters of chlorophyll a.