The purpose of this study was to assess, for the first time, the presence of muramic acid (Mur) and 3-hydroxy fatty acids (3-OH FAs), chemical markers for terrestrial bacteria in “curated” lunar samples by use of state-of-the-art gas chromatography-tandem mass spectrometry. The Apollo lunar sample collection has been stored, under isolation conditions, at the Johnson Space Center since 1969. Markers were absent in three of the four samples analyzed. However, one sample clearly contained markers for Earth bacteria (83–469 ppb for 3-OH FAs and 156 ppb for Mur). The bacterial markers were present at several orders of magnitude higher levels in terrestrial dust (7.6–36.9 × 103 ppb for 3-OH FAs and 125.3 × 103 ppb for Mur). The lunar sample containing markers consisted of dust rinsed from flight hardware, suggesting terrestrial biocontamination as the source. In conclusion, pristine lunar dust is strikingly different from terrestrial dust in lacking chemical markers for terrestrial bacteria. It is suggested that future life detection studies of other samples of extraterrestrial origin (e.g., from Mars) might be greatly aided by concurrent analysis of chemical markers for terrestrial bacteria and by including pristine lunar dust to provide a negative baseline.