The aims of this study were to prospect Penicillium and Talaromyces species associated with Austrocedrus chilensis in sites with and without Austrocedrus Root Disease (ARD), which is caused by the pathogen Phytophthora austrocedri; and to find Penicillium sp. strains with biocontrol activity against this disease. Isolations from sites with and without the prevalence of ARD were made, and the obtained isolates were phenotypically and molecularly identified to species level. Several Penicillium species were isolated from all sites; five of them were exclusively isolated from sites with ARD. Penicillium communities were compared through multivariate analyses; communities were more complex in sites with ARD. Biocontrol activity of Penicillium and Talaromyces isolates was evaluated using co-cultures with Ph. austrocedri. Penicillium glabrum, T. amestolkiae, P. palitans, and P. crustosum showed the highest biocontrol effect. Our results highlight that Penicillium species have high biocontrol activity, which also reinforces the hypothesis that some Penicillium species could be used in formulating a control strategy.