Background <p>The efficacy of biocontrol agents depends critically on their environmental adaptability, with nutrient availability being a key determinant of their success. In <i>Trichoderma</i> species, the mechanisms linking nutrient sensing to physiological responses remain poorly understood, despite their importance for biocontrol applications.</p> Results <p>This study reveals how different nitrogen sources fundamentally regulate the physiology and metabolism of <i>Trichoderma harzianum</i> through pH modulation. Under nutrient-rich conditions, <i>T. harzianum</i> exhibited a biphasic pH response characterized by initial acidification followed by alkalinization, which correlated with enhanced sporulation. Examining specific nitrogen sources, we found that sodium nitrate induced environmental alkalinization, while ammonium nitrate caused sustained acidification. These pH changes were linked to distinct physiological responses: alkaline conditions promoted sporulation, while acidic conditions enhanced mycelial growth and triggered specific metabolic responses. Notably, acidic conditions specifically induced the production of harzianic acid and related bioactive compounds, suggesting pH-dependent regulation of secondary metabolism. This nitrogen-dependent pH modulation pattern was conserved across <i>Trichoderma</i> species, as demonstrated by parallel responses in <i>T. asperellum</i>.</p> Conclusions <p>Our findings establish nitrogen source availability as a master regulator of <i>Trichoderma</i> physiology through pH-dependent mechanisms, controlling both development and secondary metabolism. This understanding provides new strategies for optimizing biocontrol formulations by manipulating nitrogen sources and pH conditions to enhance both fungal fitness and beneficial metabolite production.</p> Graphical Abstract <p></p>