Plants live in association with a multitude of microorganisms, some of which may improve the plant’s defense toward herbivores. In a previous study, we showed that Metarhizium brunneum, a fungus mostly known as an insect pathogen, can associate with cabbage roots and prime an increased myrosinase activity upon attack by Plutella xylostella larvae. Here, we ask whether another Metarhizium species, M. robertsii, also primes plant defense and whether this involves leaf glucosinolate content in addition to myrosinase activity. In addition, we tested whether priming reverses after removal of larvae. M. robertsii established and could be reisolated from roots at the end of the experiment, 43 days after inoculation. Before larval herbivory, leaf glucosinolate content and myrosinase activity did not differ between fungal-inoculated and control plants. Immediately after herbivory, in contrast, both the glucosinolate content and myrosinase activity were higher in inoculated plants than in control plants and less leaf material was consumed of the inoculated plants. Twenty-four hours after herbivory, glucosinolates, and myrosinase activity had decreased again to levels like before herbivory. Total biomass and shoot–root ratio were not affected by M. robertsii colonization. In conclusion, Metarhizium fungi can associate with Brassica roots and prime above-ground glucosinolate content and myrosinase activity. The increased defense levels were down-regulated 24 h after herbivory. Root-associated Metarhizium species may thus affect above-ground herbivores indirectly by host plant priming, in addition to their well-known direct effects as insect pathogens.