The zoophytophagous mirid bug Macrolophus caliginosus is an important biocontrol agent of whiteflies in the Mediterranean region. Periods of low productivity in commercial breeding units and unsuccessful establishment in greenhouses prompted this study of mating behaviour. Here we describe copulation behaviour, the diel mating periodicity and post-copulatory refractory period. A natural plant substrate needs to be provided if one wants to observe the copulatory behaviour of M. caliginosus in the laboratory. There was no apparent directional orientation in the approach of the two sexes, instead males pursued females after “accidentally” contacting them. Males mounted females from above, very rapidly, and without any obvious courtship behaviour, and copulation duration was very consistent (286.33 ± 4.23 s, mean ± SE). Observations over a 24-h period showed that mating was most frequent in the 8-h scotophase and first half of the 16-h photophase than in the second half of the photophase. Mated females became strongly unreceptive to new male mounting attempts, shaking their abdomen and leaving the plant if harassed. To determine the duration of the post-mating refractory period mating receptivity of females that had mated 1 or 2 weeks earlier was compared with that of virgin females of similar age. Mated females remained unreceptive even 2 weeks after mating, whereas half of the virgin females of equivalent age mated. Mated females were more likely to abandon a plant than virgin females when harassed by a male. Most males remated a few minutes after mating for the first time. M. caliginosus is atypical among mirids in that females apparently mate only once.