His style is defined by extreme precision and a fascination with the small details of nature. He is known for his series titled Archetypes and Studies. These works, such as the plate Death is the line that marks the end of all, depict animals and plants with scientific accuracy. He often used the trompe-l'oeil technique. This made his subjects appear as if they were resting on top of the paper. He frequently added insects and small mice to his compositions. These elements were often symbols of the brief nature of life. His work is a link between old manuscript traditions and new scientific inquiry.

Modern collectors value Hoefnagel for his ability to combine beauty with intellectual depth. His prints bring a sense of the Renaissance cabinet of curiosities into the home. The clean lines and white backgrounds of his botanical and zoological studies work well in contemporary interiors. They offer a quiet, focused look at nature. His art is popular with those who appreciate history and natural science.