Many biotechnological processes pursuing sustainability aim for effective, inexpensive, and environmentally friendly alternatives to replace conventional practices. Laccase-containing lignocellulolytic systems from white rot fungi have been shown to be an efficient enzymatic tool for ecofriendly biological treatments. One objective of the biotechnological enzymes production process is to find optimum growing and secretion conditions for a selected fungus. In this work, different fungi isolated from the Misiones rainforest (Coriolus versicolor f. antarcticus BAFC-266, Ganoderma applanatum strain F, Phlebia brevispora BAFC-633, and Pycnoporus sanguineus BAFC-2126) were incubated at different temperatures (25, 29, 33 °C) and pH values (3.5, 4.5, 5.5) under static conditions for 7, 10, and 14 days to evaluate their growing ability and laccase (Lac) production. Results revealed specific favorable conditions for growth and protein secretion depending on the fungus under consideration, making it necessary to adjust these parameters for each particular case. The combined effect of these cultivation parameters showed a marked influence on the secreted Lac activity by P. brevispora BAFC 633, with the highest activity (~ 240 U/l) at 29 ºC and pH 4.5 at the 10th day of cultivation. The presence of Lac isoenzymes also depended on the pH, temperature, and time of cultivation for the different tested fungi.