The 19–49 My-old obligate mutualism between Macrotermitinae termites and the Termitomyces fungus is an example of an ancient agriculture system in which Termitomyces is cultivated by termites for nutrition. Termites, in turn, keep other parasitic fungi like the parasitic weed Pseudoxylaria at bay. Unraveling the proximate mechanisms used in fungal cultivar protection is central to understanding the evolutionary stability of these farming mutualisms. We investigated the role of abiotic factors, antifungal chemicals and hygienic behaviours used by termites to keep their fungal gardens free from such parasitic fungi. Our results show the important role of abiotic factors such as termite mound temperature and CO2 in decreasing parasitic fungus growth. Using novel assays we also found that termites can display a differential behavioural response towards mutualistic and parasitic fungi and that this behaviour is coupled with antifungal activity. These results not only shed new light on how the ecology of these fungi is affected by their host but also reveal the mechanistic basis that may contribute fundamentally to the evolutionary stability of this ancient mutualism.