The past decade has seen the advent of a new imaging technique in the life sciences that is ideal for microbiological applications: the helium ion microscope (HIM). Its lateral resolution is better than one nanometre, it has a large depth-of-field and can image non-conductive specimens which renders the tool ideal for studying microbiological objects such as microbes attached to surfaces, microbial biofilms and viruses. Here we compare HIM with electron microscopy techniques and highlight selected examples that demonstrate the new possibilities for microbiology opened up by this technique.