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Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC 50 by evaluating conidia germination (38μM). To ensure high cell integrity, a sublethal dose of MND (half of LC 50 ) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC 50 and LT 50 , particularly at 10 8 conidia mL -1 . The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC 50 ), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species., (Copyright © 2021 Elsevier GmbH. All rights reserved.)